CN110470753A - Four kinds of immunosuppressor detection methods and detection kit in a kind of dry blood cake - Google Patents
Four kinds of immunosuppressor detection methods and detection kit in a kind of dry blood cake Download PDFInfo
- Publication number
- CN110470753A CN110470753A CN201910671899.2A CN201910671899A CN110470753A CN 110470753 A CN110470753 A CN 110470753A CN 201910671899 A CN201910671899 A CN 201910671899A CN 110470753 A CN110470753 A CN 110470753A
- Authority
- CN
- China
- Prior art keywords
- immunosuppressor
- quality
- control product
- dry blood
- kinds
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
Abstract
The present invention provides four kinds of immunosuppressor detection methods and detection kits in a kind of dry blood cake, wherein, four kinds of immunosuppressor detection kits in dry blood cake, including calibration object, quality-control product, internal standard mixed liquor, the first extracting solution, the second extracting solution, redissolve liquid, mobile phase A, Mobile phase B and wash needle liquid.Four kinds of immunosuppressor detection kits in dry blood cake of the present invention are capable of providing reagent involved in calibration object, sample pre-treatments and quality-control product, exempt tedious steps error with caused by of laboratory testing autogamy, guarantee the accuracy of testing result.
Description
Technical field
The invention belongs to clinical vitro detection reagent technique fields, more particularly, to four kinds of immunosupress in a kind of dry blood cake
Agent detection method and detection kit can be used for Ciclosporin A, sirolimus, tacrolimus, the Yi Weimo in dry blood cake
The detection of department.
Background technique
Immunosuppressor is the inhibited drug of the immune response to body, can inhibit and be immunoreacted related
The proliferation and function of cell reduce antibody mediated immunity reaction.Immunosuppressor is mainly used for the anti-rejection of organ transplant and itself
Immunological disease.The drug that majority acts on immune system has positive and negative both sides to act on, i.e. Immune-enhancing effect and immunosupress.It is immune
Inhibitor provides effective treatment in clinic for the rejection after autoimmunity disease and organ transplant, is widely closed
Note.
The research and development of Ciclosporin A, sirolimus, tacrolimus, everolimus, make organ transplant and autoimmune disease
Treatment reach a new high degree.But immunosupress dosage number can also cause to varying degrees the organs such as liver kidney generate
The clinical manifestation of toxic reaction, the toxic reaction and organ transplant rejection of drug is not easy to identify, and effective concentration and poisoning are dense
Degree is close.Therefore, it when carrying out immune suppressant drug treatment, needs to carry out therapeutic drug monitoring.
Currently, clinically mostly using immunization to carry out therapeutic drug monitoring, the disadvantage is that cross reaction, specificity easily occurs
Difference, different immunoassay results are not necessarily unified.With the development of technology, using chromatography as separation means, triple quadrupole bar
Tandem mass spectrum becomes trace target analytes in quantitative detection biological sample as the chromatograph-mass spectrometer coupling technology of detection means
Strong means.The existing quantitative detection side based on immunosuppressor in Liquid Chromatography-Tandem Mass Spectrometry technology detection whole blood
Fado will cause the detection knot between room since each laboratory testing method and reagent selection are different from construction method for laboratory
Fruit difference is big, especially sample is largely being detected in face of clinical, it is difficult to ensure that the consistency of testing result.Want to solve these
Problem, it is necessary to the testing process of the matched reagent box by verifying and standard be directly provided for detection project, improve liquid
Phase chromatographic tandem mass spectrum guarantees the consistency and accuracy of testing result in the generalization of clinical detection.
In addition, in the detection process, dry blood cake has that blood sampling volume is few, preparation manipulation is simple, bio-hazard is low, biological steady
Qualitative good, transport, which saves, the advantages such as facilitates, and can be preferably applied for clinic, not only reduce the demand to sample size, reduce
The physiological load of patient, and detected convenient for strange land, detection is accurate.And dry blood is carried out currently based on Liquid Chromatography-Tandem Mass Spectrometry
The detection method of four kinds of immunosuppressor and detection kit are also rarely reported in spot.
Summary of the invention
In view of this, the present invention is directed to propose four kinds of immunosuppressor detection kits in a kind of dry blood cake, existing to overcome
There is the defect of technology, be capable of providing reagent involved in calibration object, sample pre-treatments and quality-control product, exempts laboratory testing autogamy
Tedious steps and caused by error, guarantee the accuracy of testing result.
In order to achieve the above objectives, the technical scheme of the present invention is realized as follows:
Four kinds of immunosuppressor detection kits in a kind of dry blood cake, including calibration object, quality-control product, internal standard mixed liquor,
One extracting solution, the second extracting solution redissolve liquid, mobile phase A, Mobile phase B and wash needle liquid.
Preferably, the calibration object is the dry blood cake containing gradient concentration immunosuppressor.
It is furthermore preferred that the calibration object the preparation method comprises the following steps: first with methanol dilution immunosuppressor standard items it is dense to gradient
Degree, concentration range is respectively Ciclosporin A 20-1000ng/mL, sirolimus 2-100ng/mL, tacrolimus 2-100ng/
ML, tacrolimus 2-100ng/mL, are then uniformly mixed with healthy whole blood, are added drop-wise on dry blood cake and dry encapsulation.
Preferably, the quality-control product is the dry blood cake containing a certain concentration immunosuppressor；The quality-control product includes first
Quality-control product, the second quality-control product and third quality-control product, and three's concentration successively reduces.
Preferably, the content range of immunosuppressor is respectively as follows: Ciclosporin A 800- in first quality-control product
1000ng/mL, sirolimus 80-100ng/mL, tacrolimus 80-100ng/mL, everolimus 80-100ng/mL；
The content range of immunosuppressor is respectively as follows: Ciclosporin A 300-500ng/mL in second quality-control product, west
Luo Mosi 30-50ng/mL, tacrolimus 30-50ng/mL, everolimus 30-50ng/mL；
The content range of immunosuppressor is respectively as follows: Ciclosporin A 20-100ng/mL, western sieve in the third quality-control product
Do not take charge of 2-10ng/mL, tacrolimus 2-10ng/mL, everolimus 2-10ng/mL.
Preferably, the quality-control product the preparation method comprises the following steps: with methanol respectively prepare have the first quality-control product, the second quality-control product
With the standard items of third quality-control product respective concentration, corresponding standard items are uniformly mixed with healthy whole blood then, are added drop-wise to dry blood
Encapsulation is dried on spot to get the first quality-control product, the second quality-control product and third quality-control product.
Preferably, the internal standard mixed liquor is sirolimus-d3, tacrolimus-d4With the methanol solution of ascosin.
Preferably, sirolimus-d3, tacrolimus-d4It is respectively with concentration range of the ascosin in internal standard mixed liquor
300-600ng/mL、100-300ng/mL、100-300ng/mL。
Preferably, first extracting solution is methanol and the acetonitrile mixed solution of 1:1 mixing by volume.
Preferably, second extracting solution is the dilute hydrochloric acid of 0.05mol/mL, and preparation method is by hydrochloric acid distilled water
Being diluted to concentration is 0.05mol/mL.
Preferably, the redissolution liquid is methanol.
Preferably, mobile phase A is the aqueous solution containing 0.1% formic acid -2mM ammonium acetate, preparation method are as follows: weigh
0.15417g ammonium acetate solid is dissolved in a certain amount of water, and the formic acid of 1mL is added, and is settled to 1L with water.
Preferably, Mobile phase B is the methanol solution containing 0.1% formic acid -2mM ammonium acetate, preparation method are as follows: preparation side
Method is to weigh 0.15417g ammonium acetate solid to be dissolved in a certain amount of methanol, the formic acid of 1mL is added, with methanol constant volume to 1L.
Preferably, the mixed solution for washing the 1:1 mixing by volume of needle liquid first alcohol and water.
Another object of the present invention is to propose a kind of detection method of four kinds of immunosuppressor in dry blood cake, with using upper
Kit is stated, the detection of four kinds of immunosuppressor in dry blood cake is carried out with the combination of liquid chromatography-tandem level four bars mass spectroscopy device.
In order to achieve the above objectives, the technical scheme of the present invention is realized as follows:
The detection method of four kinds of immunosuppressor in a kind of dry blood cake, using kit as described above and liquid chromatogram-
QQ-TOF mass spectrometry equipment is combined the detection for carrying out four kinds of immunosuppressor in dry blood cake.
Preferably, in the dry blood cake four kinds of immunosuppressor detection method, including entire blood is removed from dry blood cake
Class seals the blood class and the first extracting solution, the second extracting solution, internal standard mixed liquor vortex mixed, and under conditions of 40-60 DEG C
The step of ultrasonic water bath is hatched；Sample will be hatched in -25 DEG C -- 15 DEG C of freezing 5-30min, and in the step of 4-10 DEG C of refrigerated centrifuge
Suddenly.
Preferably, in the dry blood cake four kinds of immunosuppressor detection method, it is characterised in that: the following steps are included:
(1) sample preparation: drawing a certain amount of whole blood and drip on filter paper, and room temperature, which is dried, to be placed in plastic sealing bag, freezing
It stores for future use；
(2) sample process: removing entire blood class from dry blood cake, by the blood class and the first extracting solution, the second extracting solution, interior
The sealing of mixed liquor vortex mixed, and the ultrasonic water bath under conditions of 40-60 DEG C are marked, 20-40min is hatched；Sample will be hatched in -25
DEG C -- 15 DEG C of freezing 5-30min, and in 4-10 DEG C of refrigerated centrifuge, it takes supernatant to carry out nitrogen and blows, then redissolve sample introduction with liquid is redissolved, use
Liquid chromatography-tandem level four bars Mass Spectrometer Method；
(3) testing result calculates: standard items being gone out peak area and go out the ratio of peak area as ordinate, mark with corresponding internal standard
The concentration of quasi- product solution obtains Ciclosporin A, sirolimus, tacrolimus, everolimus as abscissa, linear equation
Standard curve；The peak area of quality-control product sample is brought into standard curve again, actual concentration is calculated.
Preferably, in the dry blood cake four kinds of immunosuppressor detection method, chromatographic condition are as follows: mobile phase A is
The aqueous solution of 0.1% formic acid -2mM ammonium acetate, Mobile phase B are the methanol solution of 0.1% formic acid -2mM ammonium acetate；Chromatographic column is
Shim-pack GIST-HP C18 100mm × 2.1mm, 3 μm；Column temperature is 60 DEG C；Detection time is 4.5min；Sample volume is 10
μL；Condition of gradient elution: 0~0.4min, 40%B, 0.4~2.2min, 100%B, 2.2~3min, 100%B, 3~
3.5min, 40%B, 3.5~4min, 40%B, 0.5~1mL/min of flow velocity；
Preferably, in the dry blood cake four kinds of immunosuppressor detection method, Mass Spectrometry Conditions are as follows: positive ion mode,
Using the source ESI, collision gas 3Psi, gas curtain gas 25psi, atomization gas 70psi, auxiliary heats gas 55psi, spray voltage 4500V, mist
Change 300 DEG C of temperature.
Compared with the existing technology, in a kind of dry blood cake of the present invention four kinds of immunosuppressor detection kits have with
Lower advantage:
(1) it is capable of providing calibration object, the working curve for the detection of each batch sample is formulated, and exempts laboratory testing certainly
The tedious steps matched error with caused by.
(2) it is capable of providing reagent involved in sample pre-treatments and pre-treatment operating process, realizes four kinds of immunosupress
Agent avoids interference of other substances to determinand in sample from the extraction and concentration in dry blood cake.
(3) it is capable of providing quality-control product, is detected with each detection batch, guarantees the accuracy of testing result.
The detection method of four kinds of immunosuppressor has the advantage that in a kind of dry blood cake of the present invention
(1) dry blood cake has that blood sampling volume is few, and preparation manipulation is simple, reduces the demand to sample size, reduces patient's
Physiological load.
(2) bio-hazard is low, biological stability is good, transport saves conveniently, convenient for strange land detection.
Detailed description of the invention
Fig. 1 is that embodiment 1 surveys sample chromatogram figure；
Fig. 2 is the working curve diagram of Ciclosporin A in embodiment 1；
Fig. 3 is the working curve diagram of 1 sirolimus of embodiment；
Fig. 4 is the working curve diagram of tacrolimus in embodiment 1；
Fig. 5 is the working curve diagram of everolimus in embodiment 1.
Specific embodiment
In addition to being defined, technical term used in following embodiment has universal with those skilled in the art of the invention
The identical meanings of understanding.Test reagent used in following embodiment is unless otherwise specified conventional biochemical reagent；It is described
Experimental method is unless otherwise specified conventional method.
Below with reference to examples and drawings, the present invention will be described in detail.
Four kinds of immunosuppressor refer to Ciclosporin A (CsA), sirolimus (SIR), Ta Kemo in dry blood cake in the present invention
Take charge of (TAC), everolimus (EVE).
One, reagent source
(1) chemical (TRC) company reagent, CAS 59865- cyclosporin A (CsA): are studied using Toronto
13-3 is powdered reagent, and 25mg/ bottles, redissolved with methanol can be used before use.
(2) chemical (TRC) company reagent, CAS 53123-88- sirolimus (SIR): are studied using Toronto
9, it is powdered reagent, 1mg/ bottles, redissolved with methanol can be used before use.
(3) sirolimus-d3(SIR-d3): chemical (TRC) company reagent is studied using Toronto, No. CAS
162635-04-3 is powdered reagent, and 1mg/ bottles, redissolved with methanol can be used before use.
(4) chemical (TRC) company reagent, CAS 104987- tacrolimus (TAC): are studied using Toronto
11-3 is powdered reagent, and 5mg/ bottles, redissolved with methanol can be used before use.
(5) chemical (TRC) company reagent, CAS 11011-38- ascosin (Asc): are studied using Toronto
4, it is powdered reagent, 1mg/ bottles, redissolved with methanol can be used before use.
(6) chemical (TRC) company reagent, CAS 159351- everolimus (EVE): are studied using Toronto
69-6 is powdered reagent, and 5mg/ bottles, redissolved with methanol can be used before use.
(7) everolimus-d4(EVE-d4): chemical (TRC) company reagent is studied using Toronto, No. CAS
1338452-54-2 is powdered reagent, and 1mg/ bottles, redissolved with methanol can be used before use.
(8) methanol: using Fisher Chemical company 67-56-1 reagent, and purity is LC/MS grades, room temperature sealing storage
It deposits.
(9) acetonitrile: using Fisher Chemical company 75-05-8 reagent, and purity is LC/MS grades, room temperature sealing storage
It deposits.
(10) formic acid: using Fisher Chemical company 64-18-6 reagent, and purity is LC/MS grades, room temperature sealing
Storage.
(11) hydrochloric acid: using Sigma-Alorich company 7647-01-0 reagent, and purity is LC/MS grades, room temperature sealing
Storage.
(12) ammonium acetate: using Fisher Chemical company 631-61-8 reagent, and purity is LC/MS grades, and room temperature is close
Envelope storage.
(13) water: meet the level-one water of national standard GB/T 6682-2008.
Two, embodiment
The detection kit of four kinds of immunosuppressor in dry blood cake, including internal standard mixed liquor, extraction are provided in the present embodiment
Liquid 1, extracting solution 2 redissolve liquid, mobile phase A, Mobile phase B and wash needle liquid.
The calibration object solution of four kinds of immunosuppressor is configured, concentration range is respectively 20-1000ng/mL (CsA)；2~
100ng/mL (SIR, TAC, EVE) can use gradient concentration as shown in Table 1 specific to the present embodiment.
The calibration object solution gradient concentration distribution of 1 four kinds of immunosuppressor of table
| Dry blood cake biological standard curve sample | S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 |
| CsA(ng/mL) | 20 | 40 | 80 | 100 | 160 | 320 | 800 | 1000 |
| SIR(ng/mL) | 2 | 4 | 8 | 10 | 16 | 32 | 80 | 100 |
| TAC(ng/mL) | 2 | 4 | 8 | 10 | 16 | 32 | 80 | 100 |
| Eve(ng/mL) | 2 | 4 | 8 | 10 | 16 | 32 | 80 | 100 |
Quality-control product sample: containing there are four types of the dry blood cake samples of basic, normal, high three concentration of immunosuppressor.The matter of high concentration
Control product (namely first quality-control product) concentration of specimens is respectively 800ng/mL (CsA), 80ng/mL (SIR, TAC, EVE).Middle concentration
Quality-control product (namely second quality-control product) concentration of specimens is respectively 320ng/mL (CsA), 32ng/mL (SIR, TAC, EVE).Low concentration
Quality-control product (namely third quality-control product) concentration of specimens be respectively 40ng/mL (CsA), 4ng/mL (SIR, TAC, EVE).
Internal standard mixed solution: contain sirolimus-d3, ascosin, everolimus-d4Concentration be respectively 500ng/mL,
150ng/mL、150ng/mL。
Extracting solution 1: 1:1 is mixed by volume for methanol and acetonitrile.
The dilute hydrochloric acid of extracting solution 2:0.05mol/mL.
Redissolve liquid: methanol.
Mobile phase A: it weighs 0.15417g ammonium acetate solid and is dissolved in a certain amount of water, the formic acid of 1mL is added, with water constant volume
To 1L.
Mobile phase B: it weighs 0.15417g ammonium acetate solid and is dissolved in a certain amount of methanol, the formic acid of 1mL is added, uses methanol
It is settled to 1L.
Wash needle liquid: 1:1 is mixed first alcohol and water by volume.
Using mentioned reagent box to the detection method of four kinds of immunosuppressor in dry blood cake, comprising the following steps:
(1) sample preparation
20 μ L whole bloods to be drawn with pipettor to drip on Whatman903 filter paper, room temperature, which is dried, to be placed in plastic sealing bag ,-
20 DEG C store for future use.The preparation of sample to be tested: 20 μ L whole bloods are taken to drip on Whatman903 filter paper.
(2) sample treatment
Sample treatment is carried out using the kit, processing step is as follows, and entire blood is removed from dry blood cake with 8mm punch
Spot is placed in the centrifuge tube of 2mL, and the mixing internal standard of 2, the 10 μ L of extracting solution of 1, the 20 μ L of extracting solution of 290 μ L is added, and is vortexed and is mixed
It seals, ultrasonic water bath under conditions of 50 DEG C, and hatches 30min, then sample is transferred in -20 DEG C of environment and freezes 10min, it is cold
Freeze centrifugation 10min (4 DEG C, 14000rmp), then takes supernatant to carry out nitrogen and blow, finally redissolve sample introduction with the redissolution liquid of 80 μ L, use
Liquid chromatography-tandem level four bars Mass Spectrometer Method.
(3) detection method
It will redissolve and be detected in liquid sample introduction liquid chromatogram (Shimadzu)-series connection level Four pole mass spectrum (liking rich imaginative power) equipment.
Using multiple-reaction monitoring (MRM) scanning mode, chromatography test condition are as follows: mobile phase A is the water-soluble of 0.1% formic acid -2mM ammonium acetate
Liquid, Mobile phase B are the methanol solution of 0.1% formic acid -2mM ammonium acetate；Chromatographic column is Shim-pack GIST-HP C18 (100mm
× 2.1mm, 3 μm)；Column temperature is 60 DEG C；Detection time is 4.5min；Sample volume is 10 μ L；Using gradient elution, 0min~
0.4min；40%B；0.4min~2.2min, 100%B；2.2min~3min, 100%B；3min~3.5min, 40%B；
3.5min~4min, 40%B；Flow velocity is 0.9mL/min.Mass Spectrometry Conditions are positive ion mode, using the source ESI, collision gas 3psi,
Gas curtain gas 25psi, atomization gas 70psi, auxiliary heating gas 55psi, spray voltage 4500V, 300 DEG C of atomization temperature.Specific chemical combination
Object mass spectrometry parameters are as shown in table 2:
2 compound mass spectrometry parameters of table
| Compound | Parent ion (m/z) | Daughter ion (m/z) | Dwell Time(ms) | DP(V) | EP(V) | CE(V) | CXP(V) |
| CsA | 1219.9 | 1202.8 | 70 | 82 | 10 | 30 | 17 |
| SIR | 931.8 | 864.4 | 70 | 77 | 10 | 23 | 12 |
| SIR-d3 | 934.6 | 864.5 | 70 | 108 | 11 | 27 | 27 |
| TAC | 821.5 | 768.6 | 70 | 91 | 10 | 31 | 11 |
| Asc | 809.6 | 756.3 | 70 | 92 | 11 | 32 | 11 |
| EVE | 975.8 | 908.6 | 70 | 92 | 10 | 27 | 14 |
| EVE-d4 | 979.6 | 912.5 | 70 | 98 | 10 | 30 | 16 |
(remarks: CsA: Ciclosporin A；SIR: sirolimus；SIR-d3: deuterated sirolimus；TAC: tacrolimus；
Asc: ascosin；EVE: everolimus；EVE-d4: deuterated everolimus)
(4) testing result
Using the peak area of calibration object sample and internal standard peak area ratio as the ordinate of standard curve, calibration object concentration is made
Linear fit is carried out for abscissa, linear equation obtains four kinds of immunosuppressor (Ciclosporin A, sirolimus, Ta Kemo
Department, everolimus) standard curve, it is as follows:
The linear equation of Ciclosporin A are as follows: Y=0.114x-0.0718 (r=0.9962)
The linear equation of sirolimus are as follows: Y=0.0269x+0.0523 (r=0.9981)
The linear equation of tacrolimus are as follows: Y=0.0196x-0.00762 (r=0.9967)
The linear equation of everolimus are as follows: Y=0.0256x-0.01 (r=0.9988)
The range of linearity of Ciclosporin A is 20~1000ng/mL；The range of linearity of sirolimus is 2~100ng/mL；He
The range of linearity of Ke Mosi is 2~100ng/mL；The range of linearity of everolimus is 2~100ng/mL.
The peak area of quality-control product sample is brought into standard curve, actual concentration is calculated, and commented compared with theoretical value
Estimate.Quality-control product dried blood spot sample tests spectrogram as shown in Figure 1, Ciclosporin A linearity curve exemplary diagram is as shown in Fig. 2, Xi Luomo
Linearity curve exemplary diagram is taken charge of as shown in figure 3, tacrolimus linearity curve exemplary diagram is as shown in figure 4, everolimus linearity curve shows
Example diagram is as shown in Figure 5.
Three, compliance test result
1, kit correctness is verified
Dried blood spot sample is tested with kit described in embodiment, by the correctness of recovery of standard addition assay kit,
As a result as shown in Table 3-6:
3 CsA correctness of table
4 SIR correctness of table
5 TAC correctness of table
6 EVE correctness of table
By the result of table 3-6 it is found that the target analytes of basic, normal, high concentration are added in dry blood cake, recovery of standard addition is equal
The range for meeting 85%-115% shows that the accuracy of kit detection meets clinical demand.
2, kit precision is verified
Test dried blood spot sample with kit described in embodiment, respectively detection kit batch in, batch between and it is total accurate
Degree, the results are shown in Table 7.
7 kit of table test dried blood spot sample batch in, batch between and total precision
As shown in Table 7, batch of kit sample cyclosporin A, sirolimus, tacrolimus and everolimus
It is interior, batch between, total precision be respectively less than 15% (1.89%~12.90%), meet clinical detection demand.
The present invention is directed to above-mentioned existing detection immunosuppressor Ciclosporin A (CsA), sirolimus (SIR), Ta Kemo
The problem of taking charge of (TAC), everolimus (EVE), the application method of provided kit covers to be examined from sample process to sample introduction
It surveys, the total solution of data Quality Control, in addition, whole blood sample amount required for this programme is seldom, clinical reagent inspection can be met
The demand of survey.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (10)
1. four kinds of immunosuppressor detection kits in a kind of dry blood cake, it is characterised in that: including calibration object, quality-control product, internal standard
Mixed liquor, the second extracting solution, redissolves liquid, mobile phase A, Mobile phase B and washes needle liquid the first extracting solution；The internal standard mixed liquor is
The methanol solution of sirolimus-d3, tacrolimus-d4 and ascosin.
2. four kinds of immunosuppressor detection kits in dry blood cake according to claim 1, it is characterised in that: the calibration
Product are the dry blood cake containing gradient concentration immunosuppressor；Preferably, the calibration object the preparation method comprises the following steps: first using methanol dilution
For immunosuppressor standard items to gradient concentration, concentration range is respectively Ciclosporin A 20-1000ng/mL, sirolimus 2-
100ng/mL, tacrolimus 2-100ng/mL, tacrolimus 2-100ng/mL, are then uniformly mixed with healthy whole blood, are added drop-wise to
Encapsulation is dried on dry blood cake.
3. four kinds of immunosuppressor detection kits in dry blood cake according to claim 1, it is characterised in that: the Quality Control
Product are the dry blood cake containing a certain concentration immunosuppressor；The quality-control product includes the first quality-control product, the second quality-control product and third
Quality-control product, and three's concentration successively reduces；Preferably, the content range of immunosuppressor is respectively as follows: in first quality-control product
Ciclosporin A 800-1000ng/mL, sirolimus 80-100ng/mL, tacrolimus 80-100ng/mL, everolimus 80-
100ng/mL；
The content range of immunosuppressor is respectively as follows: Ciclosporin A 300-500ng/mL, Xi Luomo in second quality-control product
Take charge of 30-50ng/mL, tacrolimus 30-50ng/mL, everolimus 30-50ng/mL；
The content range of immunosuppressor is respectively as follows: Ciclosporin A 20-100ng/mL, sirolimus in the third quality-control product
2-10ng/mL, tacrolimus 2-10ng/mL, everolimus 2-10ng/mL.
4. four kinds of immunosuppressor detection kits in dry blood cake according to claim 3, it is characterised in that: the Quality Control
Product the preparation method comprises the following steps: being prepared respectively with methanol with the first quality-control product, the second quality-control product and third quality-control product respective concentration
Then corresponding standard items are uniformly mixed with healthy whole blood, are added drop-wise on dry blood cake and dry encapsulation to get the first matter by standard items
Control product, the second quality-control product and third quality-control product.
5. four kinds of immunosuppressor detection kits in dry blood cake according to claim 1, it is characterised in that: Xi Luomo
Department-d3, tacrolimus-d4It is respectively 300-600ng/mL, 100- with concentration range of the ascosin in internal standard mixed liquor
300ng/mL、100-300ng/mL。
6. four kinds of immunosuppressor detection kits in dry blood cake according to claim 1, it is characterised in that: described first
Extracting solution is methanol and the acetonitrile mixed solution of 1:1 mixing by volume；And/or second extracting solution is 0.05mol/mL's
Dilute hydrochloric acid；And/or the redissolution liquid is methanol；And/or mobile phase A is the aqueous solution containing 0.1% formic acid -2mM ammonium acetate；
And/or Mobile phase B is the methanol solution containing 0.1% formic acid -2mM ammonium acetate；And/or the needle liquid first alcohol and water of washing is by body
The mixed solution that product is mixed than 1:1.
7. the detection method of four kinds of immunosuppressor in a kind of dry blood cake, it is characterised in that: any using such as claim 1 to 6
Kit described in one and the combination of liquid chromatography-tandem level four bars mass spectroscopy device carry out in dry blood cake four kinds of immunosuppressor
Detection.
8. detection method according to claim 9, it is characterised in that: including removing entire blood class from dry blood cake, by this
Blood class and the first extracting solution, the second extracting solution, internal standard mixed liquor vortex mixed seal, and the ultrasonic water under conditions of 40-60 DEG C
The step of bath hatching；Will hatching sample in -25 DEG C -- 15 DEG C of freezing 5-30min, and in the 4-10 DEG C of refrigerated centrifuge the step of.
9. detection method according to claim 9, it is characterised in that: the following steps are included:
(1) sample preparation: drawing a certain amount of whole blood and drip on filter paper, and room temperature, which is dried, to be placed in plastic sealing bag, stored frozen
It is spare；
(2) sample process: removing entire blood class from dry blood cake, and the blood class and the first extracting solution, the second extracting solution, internal standard are mixed
The sealing of liquid vortex mixed, and the ultrasonic water bath under conditions of 40-60 DEG C are closed, 20-40min is hatched；Sample will be hatched in -25 DEG C --
15 DEG C of freezing 5-30min, and in 4-10 DEG C of refrigerated centrifuge, it takes supernatant to carry out nitrogen and blows, then redissolve sample introduction with liquid is redissolved, use liquid phase
Chromatography-QQ-TOF mass spectrometry detection；
(3) testing result calculates: standard items being gone out peak area and go out the ratio of peak area as ordinate, standard items with corresponding internal standard
The concentration of solution obtains the mark of Ciclosporin A, sirolimus, tacrolimus, everolimus as abscissa, linear equation
Directrix curve；The peak area of quality-control product sample is brought into standard curve again, actual concentration is calculated.
10. according to detection method described in claim 7-9 any one, it is characterised in that: chromatographic condition are as follows: mobile phase A is
The aqueous solution of 0.1% formic acid -2mM ammonium acetate, Mobile phase B are the methanol solution of 0.1% formic acid -2mM ammonium acetate；Chromatographic column is
Shim-pack GIST-HP C18 100mm × 2.1mm, 3 μm；Column temperature is 60 DEG C；Detection time is 4.5min；Sample volume is 10
μL；Condition of gradient elution: 0~0.4min, 40%B, 0.4~2.2min, 100%B, 2.2~3min, 100%B, 3~
3.5min, 40%B, 3.5~4min, 40%B, 0.5~1mL/min of flow velocity；
And/or Mass Spectrometry Conditions are as follows: positive ion mode, using the source ESI, collision gas 3Psi, gas curtain gas 25psi, atomization gas 70psi,
Auxiliary heating gas 55psi, spray voltage 4500V, 300 DEG C of atomization temperature.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910671899.2A CN110470753A (en) | 2019-07-24 | 2019-07-24 | Four kinds of immunosuppressor detection methods and detection kit in a kind of dry blood cake |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910671899.2A CN110470753A (en) | 2019-07-24 | 2019-07-24 | Four kinds of immunosuppressor detection methods and detection kit in a kind of dry blood cake |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110470753A true CN110470753A (en) | 2019-11-19 |
Family
ID=68508835
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910671899.2A Pending CN110470753A (en) | 2019-07-24 | 2019-07-24 | Four kinds of immunosuppressor detection methods and detection kit in a kind of dry blood cake |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110470753A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111812237A (en) * | 2020-07-08 | 2020-10-23 | 苏州药明泽康生物科技有限公司 | Valproic acid drug concentration detection kit and application thereof |
| CN111983244A (en) * | 2020-07-28 | 2020-11-24 | 天津国科医工科技发展有限公司 | Detection method and detection kit for four vitamins in dry blood spots |
| CN114019069A (en) * | 2021-05-25 | 2022-02-08 | 郑州安图生物工程股份有限公司 | Pretreatment reagent for detecting small molecular substances in biological sample |
| CN115078598A (en) * | 2022-05-05 | 2022-09-20 | 天津国科医工科技发展有限公司 | Kit for directly sampling and testing blood concentration sample and application |
| CN115078611A (en) * | 2022-05-06 | 2022-09-20 | 天津国科医工科技发展有限公司 | Mobile phase formula of immunosuppressant for liquid chromatography-mass spectrometry and detection method |
| CN115128178A (en) * | 2021-08-05 | 2022-09-30 | 上海体育学院 | Full-automatic detection method for multiple steroid esters in dried blood spot sample |
| CN115236216A (en) * | 2022-06-07 | 2022-10-25 | 合肥和合医疗科技有限公司 | Kit for detecting immunosuppressant in whole blood by high performance liquid chromatography tandem mass spectrometry, preparation method and detection method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20040087666A (en) * | 2003-04-07 | 2004-10-15 | 한국과학기술연구원 | Method for measurement of cyclosporine in blood sample using high performance liquid chromatography |
| CN102141552A (en) * | 2010-01-29 | 2011-08-03 | 中国人民解放军第二炮兵总医院 | High-sensitivity whole blood tacrolimus quantitative assay kit and preparation method thereof |
| CN109030673A (en) * | 2018-07-04 | 2018-12-18 | 易达精准（杭州）科技有限公司 | The detection method and detection kit of immunosuppressor in dried blood spot |
| CN109187839A (en) * | 2018-10-25 | 2019-01-11 | 美康生物科技股份有限公司 | The kit and detection method of four kinds of immunosuppressant drug concentrations in Accurate Determining people's whole blood |
| CN109406650A (en) * | 2018-10-25 | 2019-03-01 | 美康生物科技股份有限公司 | Kit and detection method for four kinds of immunosuppressant drug concentrations in Accurate Determining people's whole blood |
-
2019
- 2019-07-24 CN CN201910671899.2A patent/CN110470753A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20040087666A (en) * | 2003-04-07 | 2004-10-15 | 한국과학기술연구원 | Method for measurement of cyclosporine in blood sample using high performance liquid chromatography |
| CN102141552A (en) * | 2010-01-29 | 2011-08-03 | 中国人民解放军第二炮兵总医院 | High-sensitivity whole blood tacrolimus quantitative assay kit and preparation method thereof |
| CN109030673A (en) * | 2018-07-04 | 2018-12-18 | 易达精准（杭州）科技有限公司 | The detection method and detection kit of immunosuppressor in dried blood spot |
| CN109187839A (en) * | 2018-10-25 | 2019-01-11 | 美康生物科技股份有限公司 | The kit and detection method of four kinds of immunosuppressant drug concentrations in Accurate Determining people's whole blood |
| CN109406650A (en) * | 2018-10-25 | 2019-03-01 | 美康生物科技股份有限公司 | Kit and detection method for four kinds of immunosuppressant drug concentrations in Accurate Determining people's whole blood |
Non-Patent Citations (2)
| Title |
|---|
| J. C. G. DEN BURGER等: "Analysis of cyclosporin A, tacrolimus, sirolimus, and everolimus in dried blood spot samples using liquid chromatography tandem mass spectrometry", 《ANAL BIOANAL CHEM》 * |
| 李琴: "高效液相色谱-串联质谱法检测干血滤纸片上他克莫司血药浓度的研究", 《中国优秀博硕士论文全文数据库（硕士） 医药卫生科技辑》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111812237A (en) * | 2020-07-08 | 2020-10-23 | 苏州药明泽康生物科技有限公司 | Valproic acid drug concentration detection kit and application thereof |
| CN111983244A (en) * | 2020-07-28 | 2020-11-24 | 天津国科医工科技发展有限公司 | Detection method and detection kit for four vitamins in dry blood spots |
| CN114019069A (en) * | 2021-05-25 | 2022-02-08 | 郑州安图生物工程股份有限公司 | Pretreatment reagent for detecting small molecular substances in biological sample |
| CN115128178A (en) * | 2021-08-05 | 2022-09-30 | 上海体育学院 | Full-automatic detection method for multiple steroid esters in dried blood spot sample |
| CN115128178B (en) * | 2021-08-05 | 2023-08-25 | 上海体育大学 | Full-automatic detection method for various steroid esters in dried blood spot sample |
| CN115078598A (en) * | 2022-05-05 | 2022-09-20 | 天津国科医工科技发展有限公司 | Kit for directly sampling and testing blood concentration sample and application |
| CN115078611A (en) * | 2022-05-06 | 2022-09-20 | 天津国科医工科技发展有限公司 | Mobile phase formula of immunosuppressant for liquid chromatography-mass spectrometry and detection method |
| CN115236216A (en) * | 2022-06-07 | 2022-10-25 | 合肥和合医疗科技有限公司 | Kit for detecting immunosuppressant in whole blood by high performance liquid chromatography tandem mass spectrometry, preparation method and detection method thereof |
| CN115236216B (en) * | 2022-06-07 | 2024-03-01 | 合肥和合医疗科技有限公司 | Kit for detecting immunosuppressant in whole blood by high performance liquid chromatography tandem mass spectrometry, preparation method and detection method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110470753A (en) | Four kinds of immunosuppressor detection methods and detection kit in a kind of dry blood cake | |
| CN107462653B (en) | Liquid chromatography tandem mass spectrometry detection method for 7 steroid hormones in dried blood spots | |
| AU2011320345C1 (en) | LC-MS configuration for purification and detection of analytes having a broad range of hydrophobicities | |
| CN109030676A (en) | The tandem mass spectrum detection kit and its application of 16 kinds of bile acids are measured simultaneously | |
| CN109470791A (en) | A kind of method and kit of high performance liquid chromatography-tandem mass detection serum estradiol | |
| AU2007248476B2 (en) | Quantitative analysis of surface-derived samples using mass spectrometry | |
| CN109828058A (en) | Method based on a variety of liposoluble vitamins of liquid chromatography-tandem mass spectrometry | |
| CN112730706A (en) | Method for detecting biological small molecule marker by liquid chromatography-tandem mass spectrometry | |
| CN110865137A (en) | Method and kit for detecting aldosterone in blood plasma | |
| CN108693280A (en) | The method for quantitative determining the Sino-German paddy insulin content of biological sample by UPLC-MS/MS | |
| CN112964809A (en) | Kit for detecting multiple steroid hormones in biological body fluid and use method thereof | |
| Li et al. | Quantitative analysis of NIM811, a cyclophilin inhibitor, in human dried blood spots using liquid chromatography–tandem mass spectrometry | |
| CN110907569A (en) | Method for simultaneously detecting 4 protein-bound uremia toxins in blood sample | |
| CN115963199A (en) | Quantitative detection method and application of steroid hormone in human/animal body fluid | |
| CN109187814A (en) | A kind of method and its detection kit for identifying kidney transplant prognosis biomarker | |
| Wu et al. | Comparison of sample preparation methods, validation of an UPLC–MS/MS procedure for the quantification of cyclosporine A in whole blood sample | |
| CN114397379A (en) | Method for determining concentration of ornidazole in blood plasma by liquid chromatography-mass spectrometry | |
| O'Maille et al. | Metabolomics relative quantitation with mass spectrometry using chemical derivatization and isotope labeling | |
| CN112485340A (en) | Method for detecting 1, 5-sorbitan in plasma by ultra-high performance liquid chromatography tandem mass spectrometry | |
| BR112021014045A2 (en) | METHOD TO DETERMINE GLYCATED HEMOGLOBIN (HBA1C) IN A SAMPLE, KIT, KIT USE, DIAGNOSTIC SYSTEMS TO DETERMINE GLYCATED HEMOGLOBIN (HBA1C) IN A SAMPLE AND USE OF THE DIAGNOSTIC SYSTEM | |
| WO2023024727A1 (en) | Method for treating blood plasma sample containing clopidogrel oxide, and measurement method | |
| CN109406656A (en) | A kind of method and its detection kit for identifying psoriasis biomarker | |
| CN109187832A (en) | LC-MS/MS measures the method for neo-synephrine concentration and the pre-treating method of sample in blood plasma | |
| Wu et al. | Development and evaluation of a new test kit for determination of immunosuppressants in blood by UHPLC-MS/MS | |
| Sperstad | Verification of nerve agent exposure in human serum |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20191119 |
|
| RJ01 | Rejection of invention patent application after publication |